WO2021233408A1 - 抗α-溶血素的抗体及其稳定制剂 - Google Patents
抗α-溶血素的抗体及其稳定制剂 Download PDFInfo
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- WO2021233408A1 WO2021233408A1 PCT/CN2021/095074 CN2021095074W WO2021233408A1 WO 2021233408 A1 WO2021233408 A1 WO 2021233408A1 CN 2021095074 W CN2021095074 W CN 2021095074W WO 2021233408 A1 WO2021233408 A1 WO 2021233408A1
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Definitions
- the present invention relates to the field of antibody medicines. Specifically, the present invention relates to an anti- ⁇ -hemolysin antibody and its pharmaceutical use.
- Staphylococcus aureus belongs to the genus Staphylococcus. It is an important Gram-positive pathogen and the most important G + pathogen in humans. It can cause purulent infection, pneumonia, pseudomembranous enteritis, Local infections such as pericarditis, and systemic infections such as sepsis and sepsis. According to data from China Drug Resistance Surveillance Network, Staphylococcus aureus ranks 4th among pathogens detected in hospitals and 1st among G + bacteria detected in hospitals.
- Staphylococcus aureus releases a large amount of toxins to destroy tissue cells during the process of infecting the human body, and inhibits the body's immune cells to eliminate pathogens.
- ⁇ -lactam antibiotics are mainly used clinically to treat Staphylococcus aureus infections.
- antibiotics can only inhibit or kill bacteria, but cannot do anything against the toxins released by bacteria.
- bacteria will release more toxins under the pressure of antibiotics, and the lysis of bacteria killed by antibiotics will also release toxins. After a large amount of toxins enter the bloodstream, they will over-activate the host immune system, release excessive inflammatory factors, and form sepsis. disease.
- MRSA methicillin-resistant Staphylococcus aureus
- clinical treatment drugs mainly include a limited number of drugs such as vancomycin and linezolid, and these drugs are typical representatives of "super-resistant" bacteria that cause human infections. Therefore, clinically, MRSA infections are becoming more and more serious, the available antibacterial drugs are very limited, and clinical patients have failed treatment and the mortality rate remains high.
- vancomycin-resistant VRSA vancomycin-resistant Staphylococcus aureus
- hemolysin is one of the important virulence factors secreted by Staphylococcus aureus, which can be divided into four types: ⁇ -hemolysin, ⁇ -hemolysin, ⁇ -hemolysin and ⁇ -hemolysin.
- Alpha hemolysis (Hla) is a secreted toxin protein encoded by the HLA gene of Staphylococcus aureus. It is expressed in almost all strains.
- the alpha-hemolysin protein has a total length of 319 amino acids and a relative molecular mass of 33KD, which is secreted as a monomer.
- the most clear biological property of ⁇ -hemolysin is that it can quickly lyse host red blood cells and other tissue cells.
- ⁇ -hemolysin can also cause the smooth muscle contraction and convulsions of capillaries, which can lead to capillary blockage and cause ischemia and tissue necrosis.
- Staphylococcus aureus In Staphylococcus aureus, it can cause sepsis, pneumonia, breast infection, corneal infection, and Severe skin infections play an important role in the process of other diseases. At the same time, ⁇ -hemolysin can also destroy the white blood cells in the infected tissue and hinder the host from clearing the infected Staphylococcus aureus. Under the pressure of the host's immune system and antibiotics, Staphylococcus aureus at the infection site releases a large amount of ⁇ -hemolysin, which can activate the host's immune system to release excessive inflammatory factors after entering the bloodstream, resulting in sepsis.
- Antibodies are natural proteins produced by the adaptive immune system.
- the human body is given antibody drugs through passive immunization, which can not only neutralize virulence factors, but also strengthen the host's immune response to pathogens and speed up the elimination of infectious pathogens.
- ⁇ -hemolysin has shown its potential as an anti-infective antibody drug target for the treatment of Staphylococcus aureus infections: ⁇ -hemolysin is highly conserved in Staphylococcus aureus and in almost all Staphylococcus aureus Both are expressed; ⁇ -hemolysin has a clear biological function, and plays an important role in the formation of host infection and sepsis by Staphylococcus aureus; there is no homologous protein of ⁇ -hemolysin in mammalian cells, and it targets ⁇ -Hemolysin is designed for antibody drugs with low potential off-target possibility and low toxicity.
- ⁇ -hemolysin is an ideal target for anti-Staphylococcus aureus infection antibody drugs. Effective neutralization of ⁇ -hemolysin is beneficial to block the infection of Staphylococcus aureus in the human body, avoid the occurrence of sepsis after severe infection and improve the clinical The prognosis of infected patients.
- the fully human antibody R-301 developed by Aridis Pharmaceuticals with the target of ⁇ -hemolysin Has entered phase III clinical research, the main indication is severe pneumonia caused by Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus); AstraZeneca Pharmaceutical Co., Ltd.
- antibiotics such as vancomycin are still the first-line drugs for the treatment of Staphylococcus aureus infections.
- the field still needs to develop novel and efficient methods to directly neutralize toxins.
- Antibody drugs against Staphylococcus aureus In the early stage, the company developed the humanized anti-Staphylococcus aureus ⁇ -toxin antibody 78D4 H3L3 product, which can specifically bind to ⁇ -toxin and block its interaction with the receptor, thereby reducing endothelial cell damage in pneumonia. To prevent and treat pneumonia caused by Staphylococcus aureus.
- Humanized anti-Staphylococcus aureus ⁇ -toxin antibody is a biological macromolecule with a complex structure. During production and storage, physical changes such as aggregation, denaturation, precipitation, and chemical changes such as isomerization, deamidation, and oxidation occur. These changes will affect the safety and effectiveness of the product, so a stable formulation is needed to ensure that the antibody still has the biological activity required for treatment before it is used in the patient's body. At present, there is no suitable stable formulation on the market.
- the technical problem to be solved by the present invention is to provide an anti- ⁇ -hemolysin antibody molecule, especially a humanized ⁇ -hemolysin monoclonal antibody, which has the ability to bind to Staphylococcus aureus ⁇ -hemolysin and inhibit Its ability to hemolyze and damage tissue cells can be used alone or in combination with existing antibacterial drugs to treat infections or infection-related diseases caused by ⁇ -hemolysin or ⁇ -hemolysin-producing microorganisms.
- the anti- ⁇ -hemolysin antibody molecule is susceptible to changes in the physical and chemical properties of the environment during production, transportation, and storage, which in turn leads to biological activities such as ⁇ -hemolysin neutralization activity or Blocking the loss of activity; provide a stable preparation containing anti- ⁇ -hemolysin antibody, in particular, provide a stable anti- ⁇ -hemolysin antibody water injection.
- the purpose of the present invention is to provide an antibody or functional fragment thereof, and to provide its use based on the antibody or functional fragment thereof.
- the present invention provides an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein
- the heavy chain variable region includes: VH-CDR1 selected from SEQ ID NO: 22-24, VH-CDR2 selected from SEQ ID NO: 25-28, and VH shown in SEQ ID NO: 29 -CDR3;
- the light chain variable region comprises: VL-CDR1 selected from SEQ ID NO: 30-31, VL-CDR2 shown in SEQ ID NO: 32, and VL-CDR3 shown in SEQ ID NO: 33 .
- the present invention provides an antibody or fragment thereof, the antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region (VH) and the light chain variable region ( VL) respectively comprise a combination of CDRs selected from the following (VH-CDR1, VH-CDR2, VH-CDR3; VL-CDR1, VL-CDR2, VL-CDR3):
- VH-CDR1 as shown in SEQ ID NO:1, VH-CDR2 as shown in SEQ ID NO: 4, VH-CDR3 as shown in SEQ ID NO: 7; as shown in SEQ ID NO: 8 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 9 and VL-CDR3 as shown in SEQ ID NO: 10;
- VH-CDR1 as shown in SEQ ID NO: 2
- VH-CDR2 as shown in SEQ ID NO: 5
- VH-CDR3 as shown in SEQ ID NO: 7
- VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 9
- VL-CDR3 as shown in SEQ ID NO: 10;
- VH-CDR1 as shown in SEQ ID NO: 3
- VH-CDR2 as shown in SEQ ID NO: 6
- VH-CDR3 as shown in SEQ ID NO: 7
- VL-CDR1 VL-CDR2 as shown in SEQ ID NO: 9
- VL-CDR3 as shown in SEQ ID NO: 10;
- VH-CDR1 as shown in SEQ ID NO: 2
- VH-CDR2 as shown in SEQ ID NO: 6
- VH-CDR3 as shown in SEQ ID NO: 7
- SEQ ID NO: 8 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 9
- VL-CDR3 as shown in SEQ ID NO: 10;
- VH-CDR1 as shown in SEQ ID NO: 2, VH-CDR2 as shown in SEQ ID NO: 6, VH-CDR3 as shown in SEQ ID NO: 7; as shown in SEQ ID NO: 8 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 11, VL-CDR3 as shown in SEQ ID NO: 10;
- VH-CDR1 as shown in SEQ ID NO: 12, VH-CDR2 as shown in SEQ ID NO: 15 and VH-CDR3 as shown in SEQ ID NO: 18; as shown in SEQ ID NO: 19 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 20, VL-CDR3 as shown in SEQ ID NO: 21;
- VH-CDR1 as shown in SEQ ID NO: 13, VH-CDR2 as shown in SEQ ID NO: 16, VH-CDR3 as shown in SEQ ID NO: 18; as shown in SEQ ID NO: 19 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 20, VL-CDR3 as shown in SEQ ID NO: 21;
- VH-CDR1 as shown in SEQ ID NO:14, VH-CDR2 as shown in SEQ ID NO:17, VH-CDR3 as shown in SEQ ID NO:18; as shown in SEQ ID NO:19 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 20, VL-CDR3 as shown in SEQ ID NO: 21;
- VH-CDR1 as shown in SEQ ID NO: 13
- VH-CDR2 as shown in SEQ ID NO: 17
- VH-CDR3 as shown in SEQ ID NO: 18
- VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 20
- VL-CDR3 as shown in SEQ ID NO: 21;
- VH-CDR1 as shown in SEQ ID NO: 22, VH-CDR2 as shown in SEQ ID NO: 25, VH-CDR3 as shown in SEQ ID NO: 29; as shown in SEQ ID NO: 30 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 32, VL-CDR3 as shown in SEQ ID NO: 33;
- VH-CDR1 as shown in SEQ ID NO: 23, VH-CDR2 as shown in SEQ ID NO: 26, VH-CDR3 as shown in SEQ ID NO: 29; as shown in SEQ ID NO: 30 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 32, VL-CDR3 as shown in SEQ ID NO: 33;
- VH-CDR1 as shown in SEQ ID NO: 24, VH-CDR2 as shown in SEQ ID NO: 27, VH-CDR3 as shown in SEQ ID NO: 29; as shown in SEQ ID NO: 30 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 32, VL-CDR3 as shown in SEQ ID NO: 33;
- VH-CDR1 as shown in SEQ ID NO: 23, VH-CDR2 as shown in SEQ ID NO: 27, VH-CDR3 as shown in SEQ ID NO: 29; as shown in SEQ ID NO: 30 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 32, VL-CDR3 as shown in SEQ ID NO: 33;
- VH-CDR1 as shown in SEQ ID NO: 23, VH-CDR2 as shown in SEQ ID NO: 28, VH-CDR3 as shown in SEQ ID NO: 29; as shown in SEQ ID NO: 31 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 32, VL-CDR3 as shown in SEQ ID NO: 33;
- VH-CDR1 as shown in SEQ ID NO: 34, VH-CDR2 as shown in SEQ ID NO: 37, VH-CDR3 as shown in SEQ ID NO: 40; as shown in SEQ ID NO: 41 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 42 and VL-CDR3 as shown in SEQ ID NO: 43;
- VH-CDR1 as shown in SEQ ID NO: 35, VH-CDR2 as shown in SEQ ID NO: 38, VH-CDR3 as shown in SEQ ID NO: 40; as shown in SEQ ID NO: 41 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 42 and VL-CDR3 as shown in SEQ ID NO: 43;
- VH-CDR1 as shown in SEQ ID NO: 36, VH-CDR2 as shown in SEQ ID NO: 39, VH-CDR3 as shown in SEQ ID NO: 40; as shown in SEQ ID NO: 41 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 42 and VL-CDR3 as shown in SEQ ID NO: 43;
- VH-CDR1 as shown in SEQ ID NO: 35, VH-CDR2 as shown in SEQ ID NO: 39, VH-CDR3 as shown in SEQ ID NO: 40; as shown in SEQ ID NO: 41 VL-CDR1, VL-CDR2 as shown in SEQ ID NO:42, VL-CDR3 as shown in SEQ ID NO:43.
- the heavy chain variable region comprises:
- SEQ ID NO: 44 amino acid sequence or an amino acid sequence having at least 75% identity with the shown amino acid sequence;
- the light chain variable region contains:
- SEQ ID NO: 46 amino acid sequence shown in any one of ID NO: 76 or an amino acid sequence having at least 75% identity with the shown amino acid sequence.
- the heavy chain variable region and light chain variable region contained in the antibody or fragment thereof are selected from the following combinations:
- amino acid sequence shown in SEQ ID NO: 44 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 44; and, the amino acid sequence shown in SEQ ID NO: 46 Or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 46;
- amino acid sequence shown in SEQ ID NO: 48 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 48; and, the amino acid sequence shown in SEQ ID NO: 50 Or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 50;
- amino acid sequence shown in SEQ ID NO: 52 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 52; and, the amino acid sequence shown in SEQ ID NO: 54 Or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 54;
- amino acid sequence shown in SEQ ID NO: 56 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 56; and, the amino acid sequence shown in SEQ ID NO: 58 Or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 58;
- amino acid sequence shown in SEQ ID NO: 60 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 60; and, the amino acid sequence shown in SEQ ID NO: 62 Or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 62;
- amino acid sequence shown in SEQ ID NO: 72 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 72; and, the amino acid sequence shown in SEQ ID NO: 74 Or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 74;
- the aforementioned at least 75% identity is at least 80%, preferably at least 85%, more preferably at least 90%, still more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or even 99% identity and any percentage of identity ⁇ 75%.
- the antibodies or fragments thereof provided by the present invention can be monoclonal antibodies, single-chain antibodies, single-domain antibodies, bifunctional antibodies, nanobodies, fully or partially humanized antibodies or chimeric antibodies, etc.; or, the antibodies Or a fragment thereof is a half-antibody or an antigen-binding fragment of a half-antibody, such as scFv, BsFv, dsFv, (dsFv) 2 , Fab, Fab', F(ab') 2 or Fv; regarding the fragment of the antibody provided by the present invention, it is preferred Specifically, the fragment is any fragment of the antibody that can specifically bind to the antigen Staphylococcus aureus ⁇ -hemolysin.
- the antibody or antigen-binding fragment thereof of the present invention is a murine antibody, a chimeric antibody, a humanized antibody, Fab, Fab', F(ab')2, Fv, scFv.
- the antibody of the present invention is IgA, IgD, IgE, IgG or IgM, more preferably IgG1.
- the fragment of the antibody is selected from the scFv, Fab, F(ab') 2 or Fv fragment of the antibody.
- the antibody or fragment thereof further comprises a human or murine constant region, preferably a human or murine light chain constant region (CL) and/or a heavy chain constant region (CH); more preferably, the antibody or Its fragments comprise a heavy chain constant region selected from IgG, IgA, IgM, IgD or IgE and/or a kappa or lambda light chain constant region.
- the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody; more preferably, the heavy chain constant region of the monoclonal antibody is IgG1 or IgG4 Subtype, the light chain constant region is ⁇ type.
- the antibody or fragment thereof provided by the present invention comprises the heavy chain constant region shown in SEQ ID NO: 86 and/or the light chain constant region shown in SEQ ID NO: 87, or is constant with the heavy chain shown in SEQ ID NO: 86.
- the region or light chain constant region has an amino acid sequence that is at least 75% identical.
- the present invention also provides a nucleic acid molecule that encodes any antibody or fragment thereof of the present invention, or encodes the heavy chain CDR, light chain CDR, heavy chain variable region, and light chain CDR contained in the antibody or fragment thereof. Variable region, heavy chain or light chain.
- the nucleic acid molecule encodes the heavy chain variable region or the light chain variable region in the antibody or fragment thereof of the present invention.
- the nucleic acid molecule includes SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, and SEQ ID NO: 77
- SEQ ID NO: 45 includes SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ
- the present invention provides a vector comprising the nucleic acid molecule of the present invention.
- the vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, and the like.
- the vector or nucleic acid molecule of the present invention can be used to transform or transfect a host cell or enter the host cell in any manner for the purpose of preservation or expression of antibodies. Therefore, in another aspect, the present invention provides a host cell comprising the nucleic acid molecule and/or vector of the present invention, or the host cell is transformed or transfected by the nucleic acid molecule and/or vector of the present invention.
- the host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.
- the antibodies or fragments thereof, nucleic acid molecules, vectors and/or host cells provided by the present invention can be obtained by using any conventional technical methods known in the art.
- the heavy chain variable region and/or light chain variable region of the antibody can be obtained from the nucleic acid molecule provided by the present invention, or the heavy chain and/or light chain of the antibody can be obtained, and then combined with the The optional other domains of the antibody are assembled into an antibody; or, in the host cell provided by the present invention to allow the expression of the heavy chain variable region and/or light chain variable region of the antibody or the heavy chain and/or light chain of the antibody
- the host cell is cultured.
- the method further includes the step of recovering the produced antibody.
- the present invention provides a conjugate or fusion protein, which comprises the antibody or fragment thereof provided by the present invention.
- the conjugate or fusion protein may be a bispecific antibody comprising the antibody or fragment thereof of the present invention.
- the present invention provides an antibody composition comprising an anti- ⁇ -hemolysin antibody or antigen-binding fragment thereof and optional pharmaceutically acceptable excipients.
- the pharmaceutically acceptable excipients include one or more selected from the group consisting of buffers, protective agents, and surfactants.
- the concentration of anti- ⁇ -hemolysin antibody or its antigen-binding fragment is 10-100mg/mL;
- the pH of the buffer is 5.0-6.5 and the concentration is 1-50mM;
- the concentration of the protective agent is 1-10%
- the surfactant concentration is 0.001-0.1%.
- the buffer is selected from citrate buffer, histidine buffer, acetate buffer, preferably histidine buffer;
- the protective agent is selected from one or more of sucrose, trehalose, sorbitol, and mannitol, preferably sucrose;
- the surfactant is selected from Tween 20, Tween 80, and Tween 80 is preferred.
- the antibody composition of the present invention includes:
- Anti- ⁇ -hemolysin antibody or its antigen-binding fragment 30-90mg/mL
- composition of the present invention wherein
- the concentration of anti- ⁇ -hemolysin antibody or its antigen-binding fragment is 50 mg/mL;
- the concentration of histidine buffer is 10mM
- the concentration of sucrose is 5% (w/v);
- the concentration of polysorbate 80 is 0.005 to 0.015% (w/v).
- the preparation disclosed in the present invention is a water injection preparation.
- the present invention provides a stable antibody composition, which omits the anti- ⁇ -hemolysin antibody or antigen-binding fragment thereof on the basis of the antibody composition of the present invention.
- the present invention also provides the application of the composition for stabilizing the antibody in enhancing the stability of the antibody.
- the antibody in the application of the stabilized antibody composition of the present invention in enhancing the stability of the antibody, includes an anti- ⁇ -hemolysin antibody or an antigen-binding fragment thereof, preferably the anti- ⁇ -hemolysin antibody or the anti- ⁇ -hemolysin antibody of the present invention. Antigen-binding fragments.
- the antibody stability includes freeze-thaw stability, oscillation stability, and light stability.
- the present invention also provides the application of the antibody composition in the preparation of drugs for preventing or treating infections and complications caused by ⁇ -hemolysin or ⁇ -hemolysin-producing microorganisms.
- the antibodies or fragments thereof, nucleic acid molecules, vectors, host cells, conjugates or fusion proteins, etc. provided by the present invention can be included in pharmaceutical compositions, and more particularly in pharmaceutical preparations, so as to be used for each according to actual needs.
- the present invention also provides a pharmaceutical composition comprising the antibody or fragment thereof, nucleic acid molecule, vector, host cell, conjugate and/or fusion protein of the present invention, And optional pharmaceutically acceptable excipients.
- the present invention also provides a kit, which includes the antibody molecule or fragment thereof, nucleic acid molecule, vector, host cell, conjugate, fusion protein and/or pharmaceutical composition of the present invention.
- the antibody or fragments of the present invention can be used alone or in combination with other antibacterial drugs to treat or improve ⁇ -hemolysin or ⁇ -hemolysin-producing microorganisms Infection or other diseases or symptoms caused by infection. Therefore, the present invention also provides related applications of the above-mentioned themes.
- the present invention provides the use of the antibody or fragments thereof, nucleic acid molecules, vectors, host cells, conjugates, fusion proteins and/or pharmaceutical compositions of the present invention in the preparation of medicines. It is used to prevent or treat infections and complications caused by ⁇ -hemolysin or microorganisms that produce ⁇ -hemolysin.
- the present invention provides the combination of the antibody or its fragment, nucleic acid molecule, vector, host cell, conjugate, fusion protein and/or pharmaceutical composition and other antibacterial drugs or anti- ⁇ -hemolysin antibody in the preparation of medicines
- Purpose The medicine is used to prevent or treat infections and complications caused by ⁇ -hemolysin or ⁇ -hemolysin-producing microorganisms.
- the present invention provides a method for preventing or treating infections and complications caused by ⁇ -hemolysin or ⁇ -hemolysin-producing microorganisms, the method comprising administering the antibody or the antibody to a subject in need thereof Fragments, nucleic acid molecules, vectors, host cells, conjugates, fusion proteins and/or pharmaceutical compositions, and optionally antibacterial drugs.
- the optional antibacterial drug may be a drug administered in combination with the antibody or fragment thereof, nucleic acid molecule, vector, host cell, conjugate, fusion protein and/or pharmaceutical composition of the present invention.
- the combined administration of the two can take any form, including simultaneous, continuous or at intervals.
- the microorganism producing ⁇ -hemolysin is preferably Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus.
- the infection caused by ⁇ -hemolysin or ⁇ -hemolysin-producing microorganisms may be selected from upper respiratory tract infection, pneumonia, severe pneumonia, abdominal cavity infection, subcutaneous and soft tissue infection, bacteremia and various organs One or more of the infection; the complications caused by ⁇ -hemolysin or ⁇ -hemolysin-producing microorganisms may be selected from acute respiratory distress syndrome (ARDS), sepsis and body inflammatory factors One or more of high school.
- ARDS acute respiratory distress syndrome
- antibacterial drugs are drugs (including chemical drugs, biological agents and traditional Chinese medicine) that can be used to treat and prevent Staphylococcus aureus, such as methicillin-resistant Staphylococcus aureus infections, preferably antibiotics, such as ⁇ -lactam Antibiotics.
- Antibiotics can be drugs that can be used to treat methicillin-resistant Staphylococcus aureus infections listed in the guidelines/treatment strategies issued by the Infectious Diseases Society of America (IDSA, Infectious Diseases Society of America) or the Chinese Medical Association of China, preferably vancomycin , Norvancomycin, teicoplanin, linezolid, daptomycin, cefepime, fusidic acid, ceftaroline.
- the present invention also provides a method for diagnosing infections caused by ⁇ -hemolysin or ⁇ -hemolysin-producing microorganisms, the method comprising making the antibody or fragments thereof, nucleic acid molecules, vectors, host cells, conjugates, The fusion protein and/or pharmaceutical composition are contacted with a sample from the subject.
- the present invention obtains Staphylococcus aureus ⁇ -hemolysin ( ⁇ -Toxin) and non-virulent Staphylococcus aureus ⁇ -hemolysin mutein ( ⁇ -Toxin H35L) through E. coli prokaryotic expression.
- ⁇ -Toxin Staphylococcus aureus ⁇ -hemolysin
- ⁇ -Toxin H35L non-virulent Staphylococcus aureus ⁇ -hemolysin mutein
- ⁇ -Toxin H35L non-virulent Staphylococcus aureus ⁇ -hemolysin mutein
- IgG1 subtype lead antibody molecule
- the present invention uses ⁇ -Toxin H35L to successfully immunize mice to obtain spleen cells, uses hybridoma technology to establish an antibody library, and screens to obtain ⁇ -hemolysin ( ⁇ -Toxin) with high affinity and biological activity.
- ⁇ -Toxin ⁇ -hemolysin
- -Hemolysin monoclonal antibody A total of 16 lead antibody molecules were obtained, which not only have high affinity with ⁇ -hemolysin, but also have the activity of blocking ⁇ -hemolysin hemolysis.
- the present invention adopts the strategy of weak virus immunity and strong virus screening.
- the humanized antibody of the present invention can block the hemolytic effect of ⁇ -hemolysin on rabbit red blood cells and the damaging effect of ⁇ -hemolysin on lung epithelial cells in a dose-dependent manner.
- the present invention also uses the mouse ⁇ -hemolysin sepsis model, the MRSA bacteremia model and the MRSA lung infection model to evaluate the pharmacodynamics of the lead antibody molecule in animals.
- the humanized antibody of the present invention has a significant protective effect on the mouse ⁇ -hemolysin sepsis model; it can significantly prolong the survival time of the mouse MRSA bacteremia model; it can significantly reduce the mouse MRSA lungs The amount of bacteria in the infection model tissue.
- the combined application of the humanized antibody of the present invention and the commonly used antibacterial drugs vancomycin, linezolid, etc. is shown in the mouse ⁇ -hemolysin sepsis model, MRSA bacteremia model and MRSA lung infection model A significant synergistic effect.
- the animals were sacrificed and the main organs (heart, liver, spleen, lung, kidney, brain) were generally observed without abnormalities; cynomolgus monkeys were given a single dose of 10 mg/kg of the antibody of the present invention, and no animal deaths occurred. Observation for 28 days did not show any discomfort in the animals. Acute toxicity test studies show that the antibody of the present invention has good safety.
- the antibody of the present invention can effectively neutralize toxins, block its damage to the patient’s tissue cells, and at the same time improve the patient’s immunity, thereby reducing tissue damage from clinical Staphylococcus aureus infection, promoting faster clearance of infected bacteria in the patient’s body, preventing or reducing Sepsis.
- the patient can switch from intravenous infusion therapy to oral therapy more quickly, and the treatment course is shortened; at the same time, the antibody of the present invention has better clinical efficacy and tolerability, which is beneficial to the existing antibiotic therapy add.
- the antibody composition is prepared according to the physicochemical properties and biological activity of the anti- ⁇ -hemolysin antibody. Through optimized selection of buffer media, protective agents, and surfactants, the freeze-thaw stability, oscillation stability and light stability of the antibody are improved. The shelf life of antibody preparations, especially water injections, is prolonged, and the physical and chemical properties of the antibody are prevented from changing during transportation and storage, thereby losing biological activity.
- Figure 1 shows the construction of a recombinant expression plasmid of Staphylococcus aureus ⁇ -Toxin protein fused with His tag.
- Figure 2 shows the construction of a recombinant expression plasmid of the mutant ⁇ -hemolysin (H35L ⁇ -Toxin) protein of Staphylococcus aureus fused with a His tag.
- Figure 3 shows the results of 10% SDS-PAGE electrophoresis of recombinantly expressed Staphylococcus aureus mutant ⁇ -hemolysin and its mutant H35L ⁇ -Toxin, in which Figure 3A is ⁇ -Toxin, Figure 3B is H35L ⁇ -Toxin, the amount of sample It is 10 ⁇ g.
- Figure 4 shows the hemolysis of Staphylococcus aureus ⁇ -hemolysin and its mutant (H35L ⁇ -Toxin) on sheep blood plates.
- Figure 5 shows the hemolysis effect of Staphylococcus aureus ⁇ -hemolysin and its mutant (H35L ⁇ -Toxin) on rabbit blood.
- Figure 5A shows ⁇ -Toxin and
- Figure 5B shows H35L ⁇ -Toxin.
- Figure 6 shows the detection results during the screening of hybridoma cell lines.
- Figure 6A shows the ELISA detection results of antibodies in the supernatants of different cell lines and ⁇ -hemolysin
- Figure 6B shows the antibodies in the supernatants of different cell lines. Results of inhibition of ⁇ -hemolysin.
- Figure 7 shows the ELISA test results of the binding of the antibody of the present invention to ⁇ -hemolysin, wherein Figures 7A to 7D show the binding of the screened antibodies 78D4, 16H4, 78F4 and 98G9 to ⁇ -hemolysin, respectively.
- Figure 8 shows the Octect binding and dissociation curves of the antibody ⁇ -hemolysin of the present invention, wherein Figures 8A to 8D show the binding of the selected humanized versions of 78D4, 16H4, 78F4 and 98G9 antibodies to ⁇ -hemolysin, respectively .
- Fig. 9 shows the effect of the antibody of the present invention on the hemolytic activity of ⁇ -hemolysin, wherein Figs. 9A to 9C show the results when the amount of the antibody is different.
- Fig. 10 shows the therapeutic effect of the antibody of the present invention in an animal model of sepsis caused by ⁇ -hemolysin.
- Figure 11 shows the therapeutic effect of the antibody of the present invention in an animal model of bacteremia caused by methicillin-resistant Staphylococcus aureus.
- Figure 12 shows the therapeutic effect of the antibody of the present invention in an animal model of methicillin-resistant Staphylococcus aureus pneumonia.
- Figure 13 shows the results of a pharmacokinetic study of the antibody of the present invention after a single administration in cynomolgus monkeys.
- Aridis Pharmaceuticals' fully human antibody R-301 Abbreviated as AR see US9249215B2
- the variable region of the heavy chain is shown in SEQ ID NO:82
- the variable region of the light chain is shown in SEQ ID NO:83.
- the humanized antibody MEDI4893 of Astrazeneca Pharmaceuticals abbreviated as AZ (see US20140072577A1), the heavy chain variable region is shown in SEQ ID NO: 84, and the light chain variable region is shown in SEQ ID NO: 85.
- the antibody provided by the present invention has a heavy chain constant region shown in SEQ ID NO: 86 and a light chain constant region shown in SEQ ID NO: 87.
- Example 1 Recombinant expression of Staphylococcus aureus ⁇ -hemolysin ( ⁇ -Toxin) fused with His tag
- the corresponding base sequence was artificially synthesized, and it was cloned into the Pet-21a plasmid containing the His tag using the restriction sites NdeI and XhoI.
- the amino acid sequence of Staphylococcus aureus ⁇ -hemolysin is shown in SEQ ID NO: 78, and the corresponding base sequence is shown in SEQ ID NO: 79.
- the construction of the recombinant plasmid is shown in Fig. 1.
- the obtained recombinant plasmid was transformed into competent cells BL21(DE3)pLysS, and a single colony was picked the next day and inoculated into LB liquid medium containing 100 ⁇ g/ml ampicillin, and cultured overnight at 37°C with shaking.
- the overnight cultured bacteria solution was inoculated into LB liquid medium containing 100 ⁇ g/ml ampicillin at a volume ratio of 1:100, cultured with shaking at 37°C at 200 rpm to an OD 600 of approximately 0.6-0.8, and IPTG was added to the bacterial solution to the final concentration It is 0.1mM, induced at 16°C for 16-18h. Take the induced bacterial solution, centrifuge at 8,000 rpm for 3 minutes to collect the bacterial cells, and store at -80°C.
- the 35th histidine (His) was mutated to leucine (Leu) to obtain the mutated amino acid sequence, and the corresponding base sequence was artificially synthesized, and the enzyme cut position was used. Click NdeI and XhoI to clone it into Pet-21a plasmid containing His tag.
- the amino acid sequence of the mutant ⁇ -hemolysin (H35L ⁇ -Toxin) of Staphylococcus aureus is shown in SEQ ID NO: 80, and the corresponding base sequence is shown in SEQ ID NO: 81.
- the construction of the recombinant plasmid is shown in Figure 2.
- the obtained recombinant plasmid was transformed into competent cells BL21(DE3)pLysS, and a single colony was picked the next day and inoculated into LB liquid medium containing 100 ⁇ g/ml ampicillin, and cultured overnight at 37°C with shaking.
- the overnight cultured bacteria solution was inoculated into LB liquid medium containing 100 ⁇ g/ml ampicillin at a volume ratio of 1:100, and cultured with shaking at 37°C at 200 rpm to an OD 600 of approximately 0.6 to 0.8, and IPTG was added to the bacterial solution to a final concentration It is 0.25mM, induced at 25°C for 4.5h. Take the induced bacterial solution, centrifuge at 8,000 rpm for 3 minutes to collect the bacterial cells, and store at -80°C.
- the Escherichia coli that induced the expression of Staphylococcus aureus ⁇ -hemolysin and its mutants were broken with an ultrasonic disintegrator, working at 180W for 3 seconds, intermittently 3 seconds, for 7-9 minutes; centrifuged at 13,000 rpm for 30 minutes, collected the supernatant, and used a 0.22 ⁇ mL filter Filter sterilization.
- Ni column and the filtered supernatant were mixed on a rotary mixer for 1 h at room temperature, and the Ni column was loaded into the packing column.
- BB solution containing imidazole concentration of 300mM
- Figure 3 shows the electrophoresis results of the obtained protein.
- C57 mice were given different doses of Staphylococcus aureus ⁇ -hemolysin or its mutants through tail vein injection, and it was found that Staphylococcus aureus ⁇ -hemolysin was recombinantly expressed according to the methods described in Example 1, Example 2 and Example 3.
- the lowest lethal dose for C57 mice was 3 ⁇ g/mouse; the highest injection of ⁇ -hemolysin mutant was 200 ⁇ g/mouse, and the mice did not have any uncomfortable reaction.
- the ⁇ -hemolysin or ⁇ -hemolysin mutant was diluted with PBS to different concentrations, and C57 mice were injected into the tail vein. The toxic effects of the two on C57 mice were compared to select the immunogen and its dosage suitable for subsequent immunization of animals. The results are shown in Table 1.
- the Staphylococcus aureus ⁇ -hemolysin mutant was emulsified in complete or incomplete Freund's adjuvant, and injected unilaterally into three subcutaneous tissues of the back of the neck, the root of the tail, and the groin and the peritoneal cavity. Blood was collected from the tail vein on the 35th day of immunization. After the antibody titer was detected by ELISA, the spleen cells of the immunized mice were fused with myeloma cells.
- Spleen cells of Balb/c mice immunized with Staphylococcus aureus ⁇ -hemolysin mutant (H35L ⁇ -Toxin) were fused with myeloma cell P3X63Ag8.653 using PEG or electrofusion method.
- the fused hybridoma cells were seeded in 30 384-well plates, and HAT-containing medium and HT-containing medium were added 24 hours later to select hybridoma cells. After culturing in a 384-well plate for 10-14 days, take the cell supernatant and ⁇ -Toxin for ELISA experiments to screen hybridoma parent clones that can secrete antibodies that specifically bind to ⁇ -hemolysin (see Figure 6A).
- the ELISA OD value was sorted from high to low, and 94 wells of each plate were selected and transferred to 96-well plates for culture, (the 30th plate had a poorer ELISA test, and the wells were not selected for transfer). 29 96-well plates.
- the anti- ⁇ -hemolysin antibody in the cell secretion supernatant can inhibit the lysis of red blood cells by ⁇ -hemolysin.
- the degree of cell lysis by ⁇ -hemolysin and the inhibition of ⁇ -hemolysin by antibodies can be detected.
- the hemolysis test was performed on the culture supernatant of the 96-well plate after the transfer.
- the procedure is as follows: dilute WT- ⁇ -toxin hemolysin to a concentration of 5 ⁇ g/mL mother liquor, take 25 ⁇ L and mix with an equal volume of cell culture supernatant, and add it to the cell culture supernatant.
- the selected hybridoma clones secreting anti- ⁇ -hemolysin antibodies were added to a 96-well plate with feeder cells by limiting dilution method, and monoclonal cells were observed and labeled under a microscope after 2-3 days, and ELISA was performed after 7 days Experimental screening of monoclonal hybridoma cells that can secrete anti- ⁇ -hemolysin monoclonal antibodies.
- RNAfast200 kit Shanghai Feijie Biotechnology Co., Ltd.
- 5 ⁇ PrimeScript RT Master Mix Takara Reverse transcription of total RNA from hybridoma cells into cDNA
- degenerate primers Anke Krebber.1997)
- Extaq PCR reagents Takara
- PCR clean-up Gel extraction kit (Macherey-Nagel) to purify PCR amplification products; follow the instructions of pClone007 Simple Vector Kit (Qinke Biotechnology Co., Ltd.) to connect the amplified PCR products to the T vector and transform into E. coli Competent cells are amplified by strains, plasmids are extracted, and then DNA sequencing is performed to obtain monoclonal antibody variable region sequences.
- the heavy chain and light chain CDRs of the 98G9 murine antibody were defined, as shown in Table 2.
- the heavy chain and light chain CDRs of the 78F4 murine antibody were defined, as shown in Table 3.
- the heavy chain and light chain CDRs of the 78D4 murine antibody were defined, as shown in Table 4.
- the heavy chain and light chain CDRs of the 16H4 murine antibody are defined, see Table 5.
- Example 8 Combination of antibody of the present invention and ⁇ -toxin ( ⁇ -Toxin)
- the complete variable regions of the light and heavy chains of the murine antibody are combined with the constant regions of the human light and heavy chains to obtain the chimeric antibody form as a control.
- the resulting chimeric antibody was named "mouse antibody for short -xi”.
- the antigen complementarity determinant (CDR) region where the antibody binds to the antigen and the framework that supports the antibody determining the antigen complementarity determinant (CDR) region where the antibody binds to the antigen and the framework that supports the antibody’s conservative three-dimensional conformation, search for known human antibody sequences through analysis and select
- the murine antibody heavy chain CDR is inserted into the human antibody framework region to generate a humanized antibody heavy chain sequence (heavy chain version 0). Subsequently, individual amino acid positions in the mouse framework region that may be involved in antigen-antibody binding are restored to generate humanized antibody heavy chain sequences (versions 1, 2, 3).
- a humanized antibody light chain sequence (version 0, 1, 27) is generated.
- the designed and synthesized humanized antibody light and heavy chains are co-transfected into 293 cells, and the humanized antibody is expressed recombinantly (for example: heavy chain version 0 + light chain version 0 co-expression, which is H0L0, which can be further abbreviated as 00 version ).
- H0L0 heavy chain version 0 + light chain version 0 co-expression
- H0L0 which can be further abbreviated as 00 version
- the Octect instrument was used to compare the antigen binding capacity of the final humanized antibody version and the chimeric antibody xi version.
- Figure 8 shows the measurement results of some antibodies. Judging from the curve of the binding and dissociation stages of antibody and antigen binding, the specific humanized antibody version in the antigen-antibody binding and dissociation stages behaves similarly or similarly to the control antibody (including the chimeric antibody or AZ, AR antibody of the present invention). More nature.
- the sensor chip CM5 analysis channel and the control sample channel are both saturated and coupled with the maximum amount of anti-human Fc antibody, and then analyzed
- the channel flows through 7.5ug/ml of the antibody to be tested, so that the antibody is evenly distributed, and finally flows through the gradient dilution antigen sample in the analysis channel and the sample channel (starting concentration 20nM, 1:3 dilution 8 concentration points, and setting 0.741 The nm concentration point is repeated), and the light response value that occurs after the antibody antigen is bound is measured.
- the instrument software fitting (1:1) analysis the binding constant Kon and the dissociation constant Koff of the antibody, and the affinity constant KD are finally obtained.
- Example 12 Replication of an animal model of sepsis caused by ⁇ -toxin ( ⁇ -Toxin) and detection of the therapeutic effect of the antibody of the present invention
- mice were randomly divided into model control group and monoclonal antibody drug treatment group according to their body weight. Thirty minutes before the experiment, the treatment group was injected with anti- ⁇ -hemolysin monoclonal antibody (6 ⁇ g/mouse) into the tail vein, the mice in the control group were injected with the same dose of PBS, and then all mice were injected with ⁇ -hemolysin (3 ⁇ g/mouse) through the tail vein.
- ⁇ -hemolysin 3 ⁇ g/mouse
- Example 13 Replication of an animal model of bacteremia caused by methicillin-resistant Staphylococcus aureus and detection of the therapeutic effect of the antibody of the present invention
- Methicillin-resistant Staphylococcus aureus USA300 was activated on a TSB solid medium plate for 2 generations, inoculated into TSB liquid medium for overnight culture, and centrifuged at 12,000 rpm to collect the bacteria, and resuspended in physiological saline for later use.
- mice C57BL/6J mice were infected with 6 ⁇ 10 7 CFU in the tail vein of USA300 and were randomly divided into model control group (Control) and different anti- ⁇ -hemolysin monoclonal antibody drug treatment groups according to their body weight. After 2 hours of infection, the mice were injected through the tail vein. The monoclonal antibody treatment groups of each group were given 15 mg/kg of the corresponding antibody, and the control group was injected with the same dose of PBS. The survival time of the mice in each group was observed and recorded. The results are shown in Figure 11.
- Example 14 Replication of a pneumonia animal model of methicillin-resistant Staphylococcus aureus and detection of the therapeutic effect of the antibody of the present invention
- Methicillin-resistant Staphylococcus aureus USA300 was activated on a TSB solid medium plate for 2 generations, inoculated into TSB liquid medium for overnight culture, and centrifuged at 12,000 rpm to collect the bacteria, and resuspended in physiological saline for later use.
- C57BL/6J mice were infected with USA300 1.8 ⁇ 10 8 CFU/mouse through the trachea, and were randomly divided into model control group, monoclonal antibody drug treatment group, vancomycin treatment group, vancomycin + monoclonal antibody treatment group according to their body weight. After 2 hours of infection, the animals were injected through the tail vein, and the animals in each group were treated with corresponding drugs. The dosage of monoclonal antibody was 15 mg/kg, the dosage of vancomycin was 1.25 mg/kg, and the control group was injected with the same dosage of PBS. Animals were sacrificed 24 hours after infection, lung tissue homogenate was taken, weighed, homogenized, and coated on TSB solid medium to detect the amount of tissue loading. The experimental results show that the anti- ⁇ -hemolysin antibody of the present invention can enhance vancomycin treatment Pharmacodynamic effects of methicillin-resistant Staphylococcus aureus pneumonia infection.
- Example 15 Acute toxicity study of the antibody of the present invention
- mice (18-20g) are half male and half male. Each mouse is injected with 125mg/kg of 78D4 H3L3 antibody molecule through the tail vein within 24 hours. The results showed that when the dose of 125 mg/kg was administered within 24 hours, no mice died, and no discomfort was found in the animals for 14 consecutive days. The animals were sacrificed to take the main organs (heart, liver, spleen, lung, kidney, brain). ) There is no abnormality observed in general.
- Each male cynomolgus monkey was given 78D4 H3L3 antibody molecule 10mg/kg by intravenous drip in the limbs for a single dose.
- the results showed that when the 78D4 H3L3 antibody molecule was dosed at 10 mg/kg, there was no animal death, and no discomfort was observed in the animal for 28 consecutive days.
- Cynomolgus monkeys, males, each animal was given 78D4 H3L3 antibody molecule 10mg/kg via intravenous drip on the extremities, a single dose.
- 0h pre-dose, 0h
- 0.25h 15min
- 0.5h needle withdrawal point
- 4h 24h
- 48h D3
- 96h D5
- 168h D8
- 336h D15
- 504h D22
- 672h (D29) for blood sampling.
- the blood collection site is the peripheral vein of the animal's limbs (non-medicated limb) or the inguinal vein.
- the amount of blood collected is about 1 mL whole blood/mouse/time point.
- ELISA method was used to determine antibody concentration in cynomolgus monkey serum; non-compartmental model analysis method was used to calculate AUClast, CL, T1/2 and other pharmacokinetic parameters through WinNonlin Phoenix (v6.4, Pharsight) software.
- the antibody concentration of 78D4 H3L3 in the serum of cynomolgus monkeys was determined by ELISA.
- the individual graph of serum drug concentration is shown in Figure 13, and the summary of pharmacokinetic parameters is shown in Table 8. The drug exposure was seen in each animal after administration.
- Preparation method of antibody preparation Substituting the antibody into the target buffer, sub-packaging, and placing them at 4°C and 40°C for stability inspection. Samples are taken at 0 days, 7 days, and 14 days for testing.
- the test items include SEC and CEX.
- the buffer composition is as follows:
- the CEX method was used to investigate the change in the acidic peak content of 78D4 H3L3 at 40°C to determine the best buffer system and pH suitable for the antibody. The results are shown in Table 9. A straight line was fitted to the CEX acidic peak content of 0 days, 40 degrees for 7 days and 14 days at the beginning of the experiment, and the increase rate of the acid peaks (%/day) was calculated.
- the 78D4 H3L3 sample was replaced with a non-concentration buffer, and then placed at 40°C for accelerated stability experiments, and samples were taken at 0, 7, 14, and 28 days for CEX detection.
- a straight line was fitted to the CEX acidic peak content of 0 days, 40 degrees for 7 days, 14 days and 28 days at the beginning of the experiment, and the increase rate of the acid peaks (%/day) was calculated. The results are shown in Table 11.
- Table 11 shows that as the buffer concentration increases, the rate of increase of the CEX acidic peak of 78D4 H3L3 increases, and there is no significant difference between 5mM and 10mM on the CEX acidic peak. At the same time, in order to maintain the buffering capacity of the buffer, 10mM is selected as the final buffer ⁇ Liquid concentration.
- Table 12 and Table 13 show that there is no significant difference between the decrease rate of SEC purity and the increase rate of CEX acidic peak among different protective agents, so sucrose, which is commonly used in biopharmaceuticals, is selected as an additive.
- Table 14 and Table 15 show that the antibodies are relatively stable in different sucrose concentrations. Based on the principle of adding as little excipients as possible and the consideration of fully protecting the protein, 5% sucrose was selected as the protective agent for 78D4 H3L3.
- the 78D4 H3L3 antibody sample was replaced with a buffer solution of 10 mM histidine and 6% sucrose with a protein concentration of 50 mg/ml, and different final concentrations of Tween 20 and 80 were added to the replaced sample.
- the prepared samples were repeatedly frozen and thawed once, 3 times, 5 times and 10 times to determine the insoluble particles in the sample. The results are shown in Table 16 and Table 17.
- Table 16 shows that the insoluble particles in the samples without Tween 20 increased significantly, and the insoluble particles in the samples containing 0.01% Tween 20 increased significantly after freezing and thawing 10 times, indicating that 0.01% Tween 20 cannot effectively inhibit the aggregation of 78D4 H3L3 protein; ⁇ 0.02% Tween 20 has no significant difference in anti-aggregation effect.
- Table 17 shows that ⁇ 0.005% Tween 80 can effectively play an anti-aggregation effect. Compared with Tween 20, there is no significant difference in the change of insoluble particles, indicating that for the 78D4 H3L3 sample, the anti-aggregation effect of Tween 80 is better than that of Tween 20. . Therefore, 0.01% Tween 80 was selected as the surfactant of 78D4 H3L3.
- the prescription verification test includes freeze-thaw stability test, 10°C shaking stability test and light test to investigate the stability of the 78D4 H3L3 antibody in the final prescription.
- the 78D4 H3L3 protein used in the experiment was a three-step purified sample, which was concentrated to the desired concentration with a preferred antibody preparation formulation, and subjected to a shaking stability test, shaking at 10°C for 5 days. Focus on the 78D4H3L3 monomer content (SEC), charge isoform main peak content (CEX) and antibody activity (in combination with Elisa). The results are summarized in Table 18.
- the 78D4 H3L3 protein used in the experiment is a three-step purified sample, which is concentrated to the desired concentration with a preferred antibody preparation formulation, and freeze-thaw is repeated 1, 3, and 5 times. Focus on the 78D4 H3L3 monomer content (SEC), charge isoform main peak content (CEX) and antibody activity (in combination with Elisa). The results are summarized in Table 19.
- the 78D4 H3L3 protein used in the experiment is a three-step purified sample, which is concentrated to the required concentration with the preferred antibody preparation formulation, and the light stability test is carried out.
- the samples were placed under the conditions of 4500lx ⁇ 500lx for 5 days and 10 days of light, and the same samples were placed in the packaging box as a light-proof control. Focus on the 78D4 H3L3 monomer content (SEC), charge isoform main peak content (CEX) and antibody activity (in combination with Elisa). The results are summarized in Table 20.
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Abstract
Description
方法 | 重链CDR1 | 重链CDR2 | 重链CDR3 |
Chothia | SEQ ID NO:1 | SEQ ID NO:4 | SEQ ID NO:7 |
AbM | SEQ ID NO:2 | SEQ ID NO:5 | SEQ ID NO:7 |
Kabat | SEQ ID NO:3 | SEQ ID NO:6 | SEQ ID NO:7 |
组合 | SEQ ID NO:2 | SEQ ID NO:6 | SEQ ID NO:7 |
方法 | 轻链CDR1 | 轻链CDR2 | 轻链CDR3 |
Chothia | SEQ ID NO:8 | SEQ ID NO:9 | SEQ ID NO:10 |
AbM | SEQ ID NO:8 | SEQ ID NO:9 | SEQ ID NO:10 |
Kabat | SEQ ID NO:8 | SEQ ID NO:9 | SEQ ID NO:10 |
组合 | SEQ ID NO:8 | SEQ ID NO:9 | SEQ ID NO:10 |
方法 | 重链CDR1 | 重链CDR2 | 重链CDR3 |
Chothia | SEQ ID NO:12 | SEQ ID NO:15 | SEQ ID NO:18 |
AbM | SEQ ID NO:13 | SEQ ID NO:16 | SEQ ID NO:18 |
Kabat | SEQ ID NO:14 | SEQ ID NO:17 | SEQ ID NO:18 |
组合 | SEQ ID NO:13 | SEQ ID NO:17 | SEQ ID NO:18 |
方法 | 轻链CDR1 | 轻链CDR2 | 轻链CDR3 |
Chothia | SEQ ID NO:19 | SEQ ID NO:20 | SEQ ID NO:21 |
AbM | SEQ ID NO:19 | SEQ ID NO:20 | SEQ ID NO:21 |
Kabat | SEQ ID NO:19 | SEQ ID NO:20 | SEQ ID NO:21 |
组合 | SEQ ID NO:19 | SEQ ID NO:20 | SEQ ID NO:21 |
方法 | 重链CDR1 | 重链CDR2 | 重链CDR3 |
Chothia | SEQ ID NO:22 | SEQ ID NO:25 | SEQ ID NO:29 |
AbM | SEQ ID NO:23 | SEQ ID NO:26 | SEQ ID NO:29 |
Kabat | SEQ ID NO:24 | SEQ ID NO:27 | SEQ ID NO:29 |
组合 | SEQ ID NO:23 | SEQ ID NO:27 | SEQ ID NO:29 |
方法 | 轻链CDR1 | 轻链CDR2 | 轻链CDR3 |
Chothia | SEQ ID NO:30 | SEQ ID NO:32 | SEQ ID NO:33 |
AbM | SEQ ID NO:30 | SEQ ID NO:32 | SEQ ID NO:33 |
Kabat | SEQ ID NO:30 | SEQ ID NO:32 | SEQ ID NO:33 |
组合 | SEQ ID NO:30 | SEQ ID NO:32 | SEQ ID NO:33 |
方法 | 重链CDR1 | 重链CDR2 | 重链CDR3 |
Chothia | SEQ ID NO:34 | SEQ ID NO:37 | SEQ ID NO:40 |
AbM | SEQ ID NO:35 | SEQ ID NO:38 | SEQ ID NO:40 |
Kabat | SEQ ID NO:36 | SEQ ID NO:39 | SEQ ID NO:40 |
组合 | SEQ ID NO:35 | SEQ ID NO:39 | SEQ ID NO:40 |
方法 | 轻链CDR1 | 轻链CDR2 | 轻链CDR3 |
Chothia | SEQ ID NO:41 | SEQ ID NO:42 | SEQ ID NO:43 |
AbM | SEQ ID NO:41 | SEQ ID NO:42 | SEQ ID NO:43 |
Kabat | SEQ ID NO:41 | SEQ ID NO:42 | SEQ ID NO:43 |
组合 | SEQ ID NO:41 | SEQ ID NO:42 | SEQ ID NO:43 |
抗体 | ka(1/Ms) | kd(1/s) | KD(M) | Rmax(RU) | Chi2(RU2) |
78D4xiIgG | 1.40E+06 | 3.36E-04 | 2.39E-10 | 67.1 | 0.198 |
78D4-H3L3 | 1.25E+06 | 3.02E-04 | 2.40E-10 | 37.2 | 0.0303 |
98G9xiIgG | 8.84E+05 | 3.77E-04 | 4.26E-10 | 47.3 | 0.46 |
98G9-H0L2 | 7.76E+05 | 5.24E-04 | 6.74E-10 | 18.9 | 0.0677 |
98G9-H0L3 | 7.19E+05 | 5.12E-04 | 7.13E-10 | 22.5 | 0.0481 |
AZ IgG | 1.60E+06 | 2.11E-04 | 1.32E-10 | 48.8 | 0.0552 |
AR IgG | 2.76E+05 | 6.77E-05 | 2.45E-10 | 48.7 | 0.48 |
Claims (10)
- 一种抗体或其抗原结合片段,所述抗体或其抗原结合片段包含重链可变区和轻链可变区,其中所述重链可变区(VH)包含:选自SEQ ID NO:22-24之一的VH-CDR1、选自SEQ ID NO:25-28之一的VH-CDR2、以及SEQ ID NO:29所示的VH-CDR3;所述轻链可变区(VL)包含:选自SEQ ID NO:30-31之一的VL-CDR1、SEQ ID NO:32所示的VL-CDR2、以及SEQ ID NO:33所示的VL-CDR3;优选的,所述抗体或其抗原结合片段为鼠源抗体、嵌合抗体、人源化抗体、Fab、Fab'、F(ab')2、Fv、scFv。
- 一种组合物,包含权利要求1所述的抗体以及可选的药学上可接受的辅料;优选的,所述药学上可接受的辅料包括一种或多种选自缓冲液、保护剂、表面活性剂组成的组。
- 如权利要求2所述的组合物,其中,抗体或其抗原结合片段的浓度为10-100mg/mL;缓冲液的pH值为5.0-6.5、浓度为1-50mM;保护剂的浓度为1-10%;表面活性剂浓度为0.001-0.1%。
- 如权利要求3所述的组合物,其中,缓冲液选自柠檬酸缓冲液、组氨酸缓冲液、醋酸缓冲液,优选组氨酸缓冲液;保护剂选自蔗糖、海藻糖、山梨醇、甘露醇中的一种或多种,优选蔗糖;表面活性剂选自吐温20、吐温80,优选吐温80。
- 如权利要求5所述组合物,其中抗体或其抗原结合片段的浓度为50mg/mL;组氨酸缓冲液的浓度为10mM;蔗糖的浓度为5%(w/v);聚山梨醇酯80的浓度为0.005-0.015%(w/v)。
- 一种稳定抗体的组合物,其是在权利要求2-6中任一所述组合物的基础上省略了抗体或其抗原结合片段,并且所述组合物可用于增强抗α-溶血素抗体的稳定性。
- 权利要求7所述组合物在增强抗α-溶血素抗体的抗体稳定性中的应用;优选的,所述抗体稳定性包括冻融稳定性、振荡稳定性、光照稳定性。
- 权利要求2-6中任一项所述组合物在预防或治疗由α-溶血素或产生α-溶血素的微生物导致的感染及并发症中的应用,所述组合物中的抗α-溶血素抗体可以单独使用、或者与其它抗菌药物联合用于治疗由α-溶血素或产生α-溶血素的微生物导致的感染或感染相关疾病。
- 如权利要求9所述的应用,其特征在于所述其它抗菌药物包括化学药物、生物制剂和中药;优选抗生素,例如美国感染病学会(IDSA,Infectious Diseases Society of America)或者中国中华医学会颁布的指南/治疗策略中所列的可用于治疗耐甲氧西林金黄色葡萄球菌感染的药物,优选万古霉素、去甲万古霉素、替考拉宁、利奈唑胺、达托霉素、头孢吡普、夫西地酸、头孢洛林等。
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